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EP-4739776-A2 - ANTISENSE LONG NONCODING RNAS FOR THE MODIFICATION OF GENE EXPRESSION AND FOR THERAPEUTIC APPLICATIONS

EP4739776A2EP 4739776 A2EP4739776 A2EP 4739776A2EP-4739776-A2

Abstract

The present invention relates to an antisense long non-encoding RNA (aslncRNA) that may form a partial double stranded (ds) complex with a corresponding cellular mRNA in eukaryotic cells and to a biological complex comprising the antisense lncRNA and a target mRNA that may together form a partial double stranded complex in vitro. The dsRNA complex is preferentially exported from the nucleus and translated in the cytoplasm and thereby gene expression is boosted. In addition to boosting aslnc-RNAs inhibitiory aslnc RNAs are disclosed. An expression vector comprising a nucleic acid sequence of the aslncRNA is also disclosed. The present invention can be used to increase or decrease export of a target mRNA from the nucleus and thus, to increase or decrease gene expression without interfering with genomic DNA or with mRNA levels. Methods to increase or decrease the export and/or expression of a target mRNA, cell transfections using plasmids expressing the antisense lncRNA or using conjugates comprising the aslncRNA, and a pharmaceutical composition comprising the as-lncRNA are also disclosed. The aslncRNA, the complex, the expression vector, and the conjugate are useful for enhancing the production of recombinant proteins that are used in biotechnological applications and for medical purposes. The aslncRNA is also useful for increasing antigen presentation upon mRNA vaccination.

Inventors

  • KREBBER, Heike
  • BASTIANS, Holger
  • COBAN, Cesur Ivo
  • LAMPING, Jan-Philipp

Assignees

  • Georg-August-Universität Göttingen Stiftung Öffentlichen Rechts
  • Georg-August-Universität Göttingen Stiftung Öffentlichen Rechts, Universitätsmedizin

Dates

Publication Date
20260513
Application Date
20240614

Claims (20)

  1. 1. An antisense long non-encoding RNA (IncRNA) comprising a first region and a second region, wherein the first region is complementary to a portion of a target mRNA, and wherein the second region is not complementary to the target mRNA.
  2. 2. The antisense IncRNA according to claim 1, wherein the first region is complementary to the 5' end of the target mRNA.
  3. 3. The antisense IncRNA according to claim 1 or 2, wherein the second region is 3' of the first region.
  4. 4. The antisense IncRNA according to any of claims 1 to 3, wherein the antisense IncRNA is capable of forming a partial double strand complex with the mRNA thereby promoting or inhibiting the nuclear export of the mRNA.
  5. 5. The antisense IncRNA according to any one of claims 1 to 4, wherein the overlap of the first region with the 5' end of the target mRNA has a length of between 1 and thousands of nucleotides.
  6. 6. The antisense IncRNA according to any of claims 1 to 5, wherein the antisense IncRNA has a length of at least 30 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, or at least 200 nucleotides.
  7. 7. The antisense IncRNA according to any one of claims 1 to 6, wherein the antisense IncRNA is capable of forming, with the target mRNA, a complex that is optimized for binding double-strand formation mediating proteins and/or binding export mediating proteins, preferably to Dbp2, Yral, Mex67, Mtr2, or to its human homologues, comprising DDX5, ALY/REV/NXF2, TAP, pl5.
  8. 8. The antisense IncRNA according to any one of claims 1 to 7, wherein the second region has a length of between 1 and thousands of nucleotides.
  9. 9. The antisense IncRNA according to any one of claims 1 to 8, wherein the antisense IncRNA further comprises a third region that is 5' of the first region and which is not complementary to the target mRNA.
  10. 10. The antisense IncRNA according to claim 9, wherein the third region comprises between 1 and thousands of nucleotides.
  11. 11. A biological complex comprising an antisense IncRNA according to any one of claims 1 to 10 and the target mRNA, wherein the antisense IncRNA and mRNA form a partial double strand complex.
  12. 12. A biological complex comprising an antisense IncRNA according to claim 8 and the target mRNA, wherein the antisense IncRNA and mRNA form a partial double strand complex.
  13. 13. A biological complex comprising an antisense IncRNA according to any one of claims 9 or 10 and the target mRNA, wherein the antisense IncRNA and mRNA form a partial double strand complex.
  14. 14. An expression vector comprising a nucleic acid sequence encoding an antisense IncRNA according to any one of claims 1 to 10.
  15. 15. An expression vector comprising a nucleic acid sequence encoding an antisense IncRNA according to claim 8.
  16. 16. An expression vector comprising a nucleic acid sequence encoding an antisense IncRNA according to any one of claims 9 or 10.
  17. 17. An expression vector according to claim 14, further comprising a nucleic acid sequence encoding a target mRNA of the antisense IncRNA.
  18. 18. An expression vector according to claim 15, further comprising a nucleic acid sequence encoding a target mRNA of the antisense IncRNA.
  19. 19. Use of the antisense IncRNA according to claim 8, the biological complex according to claim 12, or the expression vector according to claim 15 or claim 18 for increasing export of the target mRNA from the nucleus of a eukaryotic cell via a partial double strand complex with the antisense IncRNA.
  20. 20. Use of the antisense IncRNA according to claim 8, the biological complex according to claim 12, or the expression vector according to claim 15 or claim 18 for increasing expression of the target mRNA in a eukaryotic cell by increasing nuclear export of the target mRNA via a partial double strand complex with the antisense IncRNA.

Description

ANTISENSE LONG NONCODING RNAS FOR THE MODIFICATION OF GENE EXPRESSION AND FOR THERAPEUTIC APPLICATIONS BACKGROUND OF THE INVENTION [0001] A dogma in gene expression is that mRNAs, which are generated in the nucleus, are subsequently transported into the cytoplasm where they are translated into protein. High throughput technologies have identified a large number of non-coding RNAs (ncRNAs), some of which represent antisense strands that are complementary to parts of the mRNAs. Their function, however, remained mostly elusive, but it seemed that they have important roles in cells because several reports showed that their dysregulation is associated with diseases such as cancer or neurodegenerative diseases. Such antisense (as)RNAs exist in all organisms. [0002] Typically, ncRNAs are divided into small (below 200 nucleotides) and long ncRNAs (exceeding 200 nucleotides) (IncRNAs). IncRNAs are represented globally in living organisms and are perhaps the least well-understood type of transcripts. IncRNAs share many similar characteristics to mRNA, for instance, they are transcribed by RNA polymerase II with similar chromatin states, undergo 5'-capping, splicing and 3'-polyadenlation. Although it has been described that IncRNAs contain a small open reading frame (smORF) from which functional small peptides are generated, the majority of IncRNAs lack an ORF and therefore the coding potential of mRNAs. [0003] While reports exist that speculate on potential functions for IncRNAs in regulating transcription, for example, in response to environmental changes, a general function for many IncRNAs is currently unknown. In fact, significant amounts of antisense IncRNAs travel into the cytoplasm and are eliminated through the cytoplasmic exonuclease known as Xrnl, similar to the so- called XUTs identified in yeast. Xrnl mediated degradation follows recognition through the nonsense mediated decay (NMD) system, which detects a lack of correct ORFs and has been suggested to eliminate these cytoplasmic IncRNAs. Exporting and translating such antisense (as)RNAs appears to involve undue energy consumption by the cell at first sight, since no general cytoplasmic function for these IncRNAs has yet been discovered in eukaryotes. SUMMARY OF THE INVENTION [0004] In one aspect, the present invention relates to an antisense long non-coding RNA (abbreviated herein as antisense IncRNA, asIncRNA, or IncRNA) comprising a first region and a second region, wherein the first region is complementary to a portion of a target mRNA, and wherein the second region is not complementary to the target mRNA. In an embodiment, the first region is complementary to the 5' end of the target mRNA. In another embodiment, the first region is complementary to the 5' UTR of the target mRNA. In another embodiment, the second region is 3' of the first region. In another embodiment, the antisense IncRNA is capable of forming a partial double strand complex with the mRNA thereby promoting or inhibiting the nuclear export of the mRNA. In an embodiment, the overlap of the first region with the 5' end of the target mRNA has a length of between 1 and thousands of nucleotides. In an embodiment, the complementary region has a length of between 1 and thousands of nucleotides. In another embodiment, the complementary region has a length of between 20 and 3000. In another embodiment, the complementary region has a length of between 20 and 1000. In another embodiment, the complementary region has a length of between 50 and 500. In another embodiment, the antisense IncRNA is capable of forming, with the target mRNA, a complex that is optimized for binding double-strand formation mediating proteins and/or binding export mediating proteins, preferably to Dbp2, Yral, Mex67, Mtr2, or its human homologues, DDX5, ALY/REV/NXF2, TAP, pl5. Other or additional helicases may be comprised in the complex. [0005] In an embodiment, the second region of the antisense IncRNA has a length of between 1 and thousands of nucleotides. In another embodiment, the second region has a length of between 1 and 5000. In another embodiment, the second region has a length of between 1 and 2000. In another embodiment, the second region has a length of between 1 and 800. In an embodiment, the antisense IncRNA consists of a first region and a second region. In an embodiment, the antisense IncRNA consists of a first and a second region, wherein the second region has a length of between 1 and thousands of nucleotides. In an embodiment, the antisense IncRNA consists of a first and a second region, wherein the first region is complementary to a portion of the target mRNA and has a length of between 1 and thousands of nucleotides, and wherein the second region is 3' of the first region, wherein the second region is not complementary to the mRNA and wherein the second region has a length of between 1 and thousands of nucleotides. In an embodiment, the antisense IncRNA consists of a first and a second region, where